Quinolone carboxylic acid derivatives in crystalline hydrate form

ABSTRACT

1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylic acid dihydrate having the following formula: ##STR1## The crystal of this dihydrate exhibits excellent stability over the other crystal forms under various pharmaceutical formulation conditions such as moisture absorption and blending in solvents and, hence, it is a most advantageous crystal form in the manufacture of pharmaceuticals.

This application is a 371 of PCT/JP 93/00109, filed 29 Jan. 1993.

TECHNICAL FIELD

This invention relates to1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid dihydrate that is useful as an antimicrobial agent and which hassatisfactory stability.

BACKGROUND ART

The official gazette of Japanese Patent Public Disclosure (KOKAI) No.Hei 3-95177 discloses1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid (hereunder designated "Q-35"). The official gazette further teachesthat Q-35 is the product of recrystallization from acetonitrile and thatit has satisfactory antimicrobial activity.

However, as continued research was undertaken to commercialize it as amedicine, the Q-35 recrystallized from acetonitrile turned out to haveonly low stability due to the drawback that its weight would increasewith increasing humidity. Under the circumstances, it was impossible toadminister the Q-35 in well-defined doses and this, combined with otherproblems of the Q-35 made it difficult to develop said compound as amedicine. Hence, there was the need to develop a technique by whichstable Q-35 could be produced even under humid conditions.

DISCLOSURE OF INVENTION

The present inventors conducted intensive studies with a view toeliminating the above-described drawback of Q-35 recrystallized fromacetonitrile. As a result, they found that Q-35 had four crystal forms,a crystal with a variable water content (which is hereunder referred toas "crystal III" or "type III crystal"), a monohydrate crystal (which ishereunder referred to as "crystal II" or "type II crystal"), a dihydratecrystal (which is hereunder referred to as "crystal I" or "type Icrystal"), and an anhydride crystal, and that the specific type ofcrystal to be produced is determined by the type of solvent used forrecrystallization. As a result of closer studies conducted on thephysical properties of the respective crystal forms, the inventors foundthe following: the Q-35 recrystallized from acetonitrile was a type IIIcrystal; type I crystal, namely, the dihydrate of Q-35, was the moststable under humid conditions and, although it turned to an anhydrideunder drying or heating conditions, it reverted to the dihydrate whenleft to stand. The present invention has been accomplished on the basisof this finding. Stated briefly, the invention relates to a1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid dihydrate having the following formula: ##STR2##

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the weight change of type I crystal of Q-35 when it wasstored under atmospheric conditions after heating;

FIG. 2 shows the weight change of type I crystal of Q-35 when it wasfirst stored under dried conditions at room temperature, then storedunder atmospheric conditions;

FIG. 3 shows the weight change of dehydrated type I crystal of Q-35 whenit was stored under dried conditions (6% R.H.) at room temperature;

FIG. 4 shows TG and DTA curves obtained when type I crystal of Q-35 washeated from room temperature up to 170° C. at a rate of 3° C./min;

FIG. 5 shows a DSC curve obtained when type I crystal of Q-35 was heatedfrom room temperature up to 170° C. at a rate of 3° C./min;

FIG. 6 shows infrared absorption spectra for type I crystal of Q-35 inboth the initial and heated states;

FIG. 7 shows infrared absorption spectra for type I crystal of Q-35 bothin the heated state and after cooling in an anhydrous atmospherefollowed by storage at room temperature;

FIG. 8 shows infrared absorption spectra for type I crystal of Q-35 bothin the initial state and after heating followed by storage underatmospheric conditions;

FIG. 9 shows infrared absorption spectra for type I crystal of Q-35 bothin the heated state and after storage in an anhydrous atmosphere at roomtemperature;

FIG. 10 shows infrared absorption spectra for type I crystal of Q-35both in the initial state and after storage in an anhydrous state atroom temperature followed by storage under atmospheric conditions;

FIG. 11 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the initial state;

FIG. 12 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the heated state;

FIG. 13 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 as obtained when it was heated followed by cooling under driedconditions at storage at room temperature;

FIG. 14 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 as obtained when it was heated followed by cooling under driedconditions and storage under atmospheric conditions;

FIG. 15 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the heated state;

FIG. 16 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 after storage under dried conditions at room temperature;

FIG. 17 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 as obtained when it was stored under dried conditions at roomtemperature, followed by storage under atmospheric conditions;

FIG. 18 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the initial state;

FIG. 19 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the initial state as it was placed under atmospheric conditions;

FIG. 20 is a composite spectrum for the powder X-ray diffraction of typeI crystal of Q-35 in the initial state as obtained from the result ofsingle-crystal X-ray analysis;

FIG. 21 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 in the heated state;

FIG. 22 is a composite spectrum for the powder X-ray diffraction of typeI crystal of Q-35 under dried conditions at room temperature as obtainedfrom the result of single-crystal X-ray analysis;

FIG. 23 shows the crystal structure of type I crystal of Q-35 in theinitial state;

FIG. 24 shows stereographically the crystal structure of type I crystalof Q-35 in the initial state;

FIG. 25 shows the crystal structure of type I crystal of Q-35 underdried conditions at room temperature (as a dehydrated product);

FIG. 26 shows stereographically the crystal structure of type I crystalof Q-35 under dried conditions at room temperature (as a dehydratedproduct);

FIG. 27 shows the crystal structure of type I crystal of Q-35 as storedunder dried conditions at room temperature, followed by storage underatmospheric conditions;

FIG. 28 shows stereographically the crystal structure of type I crystalof Q-35 as stored under dried conditions at room temperature, followedby storage under atmospheric conditions;

FIG. 29 shows powder X-ray diffraction spectra for type II crystal ofQ-35 as stored in humidified conditions, FIG. 29a showing a powder X-raydiffraction spectrum for the case of storage at 40° C.×0% R.H. for oneweek, FIG. 29b for storage at 40° C.×75% R.H. for one week, and FIG. 29cfor storage at 40° C.×100% R.H. for one week;

FIG. 30 is a powder X-ray diffraction spectrum for type I crystal ofQ-35 as stored at 40° C.×100% R.H. for one week;

FIG. 31 shows powder X-ray diffraction spectra for type II crystal ofQ-35 after blending, FIG. 31a showing a powder X-ray diffractionspectrum for a powder blended in ethanol, FIG. 31b for a powder blendedin an aqueous solution of 50% ethanol, and FIG. 31c for a powder blendedin water; and

FIG. 32 shows powder X-ray diffraction spectra for type I crystal ofQ-35 after blending, FIG. 32a showing a powder X-ray diffractionspectrum for a powder blended in ethanol, FIG. 32b for a powder blendedin an aqueous solution of 50% ethanol, and FIG. 32c for a powder blendedin water.

BEST MODE FOR CARRYING OUT THE INVENTION

Q-35 can be synthesized either by a method (process I) in which1-cyclopropyl-6,7-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid(DFQ) is condensed directly with 3-methylaminopiperidine (3-MAP) or by amethod (process II) in which DFQ-Et is reacted with HBF₄ to form DFQ-BF₂chelate (DFQ-BF₂), which is condensed with 3-MAP to form Q-35 BF₂chelate (Q-35-BF₂), which is thereafter hydrolyzed with Et₃ N or anaqueous solution of NaOH or the like to yield Q-35. Process II achievesa higher yield and, hence, is suitable for large-scale synthesis. Thereaction routes of processes I and II are as following: ##STR3##

For purification, the Q-35 obtained by process I or II is heated forrefluxing and drying in a solvent, then purified with a purifyingsolvent. Which of the crystal forms described hereinabove will beyielded in this case depends on the purifying solvent used. Ifacetonitrile-water is used, either type III crystal or type II crystalis obtained; with methanol, type II crystal is obtained; and withethanol-water (1:1), type I crystal is obtained. The present inventorsstudied under what conditions these three kinds of crystal forms wouldbe obtained and discovered the following: type III crystal yielded whenthe formation of a complete solution in ethanol or acetonitrile wasfollowed by distilling off the solvent under vacuum; type II crystalyielded when the formation of a suspension in methanol was followed byheating under reflux; and type I crystal yielded when the formation of asuspension in ethanol-water (1:1) was followed by heating under reflux.

Crystal II transforms to crystal I upon wetting and blending (in 50%ethanol or water). On the other hand, crystal I shifts to crystal IIupon heating under reflux in the presence of methanol but no crystaltransformation occurs upon wetting and blending (in 50% ethanol orwater). The inventors further verified that upon drying, crystal II andcrystal I were deprived of the water of crystallization to becomeanhydrides but that upon standing in air, the anhydrides reverted totheir respective hydrate forms.

Shown below are examples of the production of the compound of thepresent invention but it should be understood that the invention is inno way limited to those examples.

EXAMPLE 1

DFQ-BF₂ ester (3.4 g), 3-methylaminopiperidine.2HCl (3-MAP.2HCl; 2.3 g)and triethylamine (4.5 g) were added to methylene chloride (18 ml) andthe mixture was heated under reflux for 3 h. After distilling off themethylene chloride under vacuum, a solution consisting of NaOH (2.5 g)and water (20 ml) was added and reaction was carried out at 80° C. for1.5 h. After cooling, the reaction solution was adjusted to a pH of 8-9with 6N HCl for crystallization. The precipitating crystal wascentrifuged to give a wet powder of crude Q-35 in an amount of 4.2 g(3.2 g on a dry basis; yield, 83.0%).

Fumaric acid (3.5 g) was dissolved in a 90% aqueous methanol solution(102 ml). To the resulting solution, crude Q-35 was added in an amountof 9.4 g (on a dry basis). The solution was cooled and the precipitatingcrystal was centrifuged to give a wet powder of Q-35.fumarate in anamount of 12.1 g (11.0 g on a dry basis; yield, 90.1%).

NaOH (3.6 g) was dissolved in water (100 ml). To the resulting solution,Q-35.fumarate (11.0 g) was added and a solution was formed. Afterseparating the insolubles by filtration, 6N HCl was added to adjust thepH to 8-9 for crystallization. The precipitating crystal was centrifugedand dried to produce type I crystal of purified Q-35 in an amount of 7.7g (yield: 83.2%).

EXAMPLE 2

A 200-ml reaction vessel was charged with a 9.1% (w/w) MAP methanolsolution (61.7 g, 49.3 mmol) and heated with warm (60° C.) water undervacuum to distill off about 55 ml of methanol. To the resultingconcentrate residue, methylene chloride (65 ml) triethylamine (7.7 g,75.8 mmol) and DFQ-BF₂ ester (13.0 g, 37.9 mmol) were added and themixture was reflexed for 1 h. A solution formed gradually and it turnedclear and yellow. The solvent in the reaction solution was distilled offunder vacuum. To the concentrate residue, water (30 ml) and a 25%aqueous sodium hydroxide solution (39 g, 244 mmol) were added andhydrolysis was performed at 70° C. for 1 h (upon heating to about 50°C., the remaining solvent stated to distill off). After water-coolingthe liquid hydrolysis mixture, its pH was adjusted to 8.5 with about 30ml of 5.5N HCl (1/1) and heating was done at 60° C. for 30 min topromote crystallization. The liquid mixture was cooled to 25° C. andstirred for 1 h. Subsequently, the liquid mixture was loaded in a 24-incentrifuge for about 45 min to separate the crystal. The resultingcrystal was washed with water (20 ml) and shaken out for 30 min to givea wet powder of crude Q-35 in an amount of 18.2 g (net: 13.8 g; yield,94%).

A 200-ml reaction vessel was charged with ion exchanged water (100 ml),conc. HCl (4.3 ml, 47.3 mmol) and the wet powder of crude Q-35 (18.2 g;net=13.8 g (35.5 mmol)) to give a pH of 3-4. Two extractions wereconducted, each with 30 ml of ethyl acetate. By heating with warm (70°C.) water under vacuum, the ethyl acetate dissolved in aqueous layer wasdistilled off (ca. 1.5 h). The aqueous layer was rendered acidic byaddition of HCl (2 ml) and the resulting small amount of insolubles wereseparated by filtration. After being adjusted to a pH of 8.5 with about8 ml of a solution of sodium hydroxide in water (3 g in 10 ml), thefiltrate was heated at 60° C. for 30 min to promote crystallization.After the end of heating, the solution was cooled to 25° C. and stirredfor 1 h. Subsequently, the filtrate was loaded in a 24-in centrifuge forabout 30 min to separate the crystal. The resulting crystal was washedwith ion-exchanged water (20 ml) and shaken out for 30 min to produce13.7 g of the crystal.

A 200-ml reaction vessel was charged with ethanol (80 ml), water (80 ml)and the crystal (13.7 g) and the mixture was heated at 70° C. andstirred for 30 min as a suspension. The resulting liquid mixture wascooled to 25° C. and stirred for 1 h, followed by loading in a 24-incentrifuge for about 30 min to separate the crystal. The resultingcrystal was washed with ion-exchanged water (20 ml) and shaken out for30 min to produce a wet powder of Q-35. Using a through-flow dryer, thewet powder was dried at 60° C. for 2 h, then aerated at room temperaturefor 2 h to produce type I crystal of Q-35 in an amount of 10.1 g (yield,73%).

Using the thus obtained type I crystal of Q-35, the followingexperiments were conducted in order to unravel its structure, as well asthe behavior of the bimolecular water of crystallization.

EXPERIMENTS

1) Samples

As samples for infrared absorption spectroscopy, powder X-raydiffraction and thermal analyses, those which were produced by themethods of the Examples were used. As samples for single-crystal X-rayanalysis, those which were prepared by the method described within thefollowing parentheses were used. (Crystal for single-crystal X-rayanalysis: Absolute ethanol (450 ml) was added to type I crystal of Q-35(8.10 g) prepared by the methods of Examples 1 and 2 and the mixture washeated at 75° C. for 30 min to filter it while hot. After being left tostand at room temperature, the filtrate was further filtered by means ofsuction to produce a crystal (ca. 5.95 g). Water (300 ml) was added tothe crystal and the mixture was heated at 95° C. for 5 min; afterstanding at room temperature, the mixture was filtered by means ofsuction and the filtrate was left to stand at room temperature to yielda crystal.)

2) Apparatus Used

TG/DTA: TG/DTA 200 of Seiko Denshi K.K.

DSC: DSC 210 of Seiko Denshi K.K.

Infrared spectrophotometer: 20 DXB of Nicolet

Powder X-ray diffractometer: PW 1730/10 of Phillips

Single-crystal X-ray diffractometer: CAD4 of Enraf-Nonius

3) Experimental Methods

(1) Thermal Analyses

1 Heating and Cooling Experiment (TG)

About 10 mg of a sample (being a powder, the sample need not bepulverized) was heated from room temperature up to 80° C. at a rate of5° C/min, held at 80° C. for 30 min and thereafter cooled to roomtemperature. The changes that occurred in the weight of the sample as aresult of its heating and cooling were examined. To avoid its dryingeffect, N₂ gas was not allowed to flow during the measurement (relativehumidity in the room: 40-50 % R.H.)

2 Experiment Under Exposure to an Anhydrous Atmosphere at RoomTemperature Followed by Standing Under Atmospheric Condition (TG)

About 10 mg of a sample (not pulverized) was left to stand in ananhydrous atmosphere at room temperature with N₂ gas being allowed toflow at 200 ml/min and the resulting changes in the weight of the samplewere examined. When there was no longer a change in the sample's weight,the supply of N₂ gas was stopped to create atmospheric conditions(relative humidity in the room: 40-50% R.H.), in which the changes inthe sample's weight were examined again.

3 Experiment for Storage at Low Humidity (6% R.H.) (TG)

About 10 mg of a sample (not pulverized) was heated to dehydrate in ananhydrous atmosphere under a N₂ gas flow. Thereafter, air* humidified to6% R.H. was allowed to flow at 200 ml/min at room temperature and theresulting changes in sample's weight were examined.

4 Heating Experiment and the Calculation of Activation Energy (TG/DTA)

About 10 mg of a sample (not pulverized) was heated from roomtemperature up to 170° C. at rates of 2°, 3° and 5° C./min. Theresulting changes in sample's weight and the thermal changes thataccompanied were examined and the activation energy was determined fromthe weight changes by the Ozawa method. To avoid its drying effect, N₂gas was not allowed to flow during the measurement (relative humidity inthe room: 40-50% R.H.)

5 Heating Experiment (DSC)

About 10 mg of a sample (not pulverized) was subjected to measurements,with the sample pan being kept open with no crimps applied in order toavoid pressurization by steam. During the measurement, N₂ gas wasallowed to flow at 20 ml/min and when thermal stability was reached (inabout 3 min), the sample was heated from room temperature up to 170° C.at a rate of 3° C./min and the resulting thermal changes were examined.

(2) Infrared Absorption Spectra

1 Heating (80° C.) Followed by Standing in an Indoor Atmosphere

A sample was mixed and diluted with KBr to a concentration of 5%, heatedin a heating cell for powder X-ray diffraction and subjected to ameasurement by the method of diffuse reflection analysis (DRA); thenumber of scans, 2048; gain, 16. Both the heating experiment and theexperiment in an indoor atmosphere were conducted with the samplechamber being kept open to avoid the effect of drying air (relativehumidity in the room: 20-30% R.H.) and the same procedure was followedto perform measurements on the reference. In the experiment in ananhydrous atmosphere, the sample chamber was closed and the anhydrousatmosphere was created by supplying drying air and the same procedurewas followed to perform measurements on the reference.

2 Experiment in an Anhydrous Atmosphere at Room Temperature, Followed byExperiment in an Indoor Atmosphere

A sample was mixed and diluted with KBr to a concentration of 5% andsubjected to measurements by the method of diffusive reflection analysis(DRA); the number of scans, 1024; gain, 8. A convenient DRA cell wasused for the measurements. The experiment under dried conditions wasconducted with the sample chamber being closed under a drying air flow,and the same procedure was followed to perform measurements on thereference. In the experiment under atmospheric conditions, the samplechamber was kept open (relative humidity in the room: 20-30% R.H.) andthe same procedure was followed to perform measurements on thereference.

(3) Powder X-ray Diffraction Spectra

1 Heating (80° C.) Followed by Standing Under Atmospheric Conditions

A sample was pulverized and heated up to 80° C. in a heating cell at arate of 5° C./min; thereafter, N₂ gas was allowed to flow to create ananhydrous atmosphere, followed by cooling to room temperature.Subsequently, the supply of N₂ gas was stopped to create an indooratmosphere for measurement (relative humidity in the room: 60-70% R.H.)

2 Exposure to an Anhydrous Atmosphere at Room Temperature, Followed byStanding in an Indoor Atmosphere

A sample was pulverized and placed in a heating cell, through which N₂gas was allowed to flow to create an anhydrous atmosphere andmeasurements were performed at given time intervals. Thereafter, thesupply of N₂ gas was stopped to create atmospheric conditions andmeasurements were conducted (relative humidity in the room: 60-70%).

(4) Single-crystal X-ray Analysis

After measurements (R.H.: 60-70% at room temperature), N₂ gas wasallowed to flow to create an anhydrous atmosphere and measurements wereconducted. Thereafter, the sample was stored again under atmosphericconditions and measurements were conducted.

4) Experimental Results and Discussion

(1) Analyzing the Behavior of the Water of Crystallization by ThermalAnalyses

Type I crystal (dihydrate) of Q-35 was heated from room temperature upto 80° C. (without flowing N₂ gas so that it would not cause any adverseeffects) in the TG method; as a result, the weight of the crystaldecreased with the increasing temperature and the ultimate weight losswas about 8.1%. Since the theoretical value of the water content in typeI crystal of Q-35 is 8.47%, the weight loss is estimated to correspondto the water of crystallization. In other words, the sample after weightloss due to heating would be a dehydrated anhydride. In the subsequentcooling phase, the weight of the sample started to increase as soon asits temperature was lowered and the initial weight was restored in about150 min (FIG. 1). From these facts, it was estimated that the twomolecules of the water of crystallization in type I crystal of Q-35 wereeliminated upon heating but that the crystal incorporated the moistureof air at room temperature to become stabilized again in the state ofbimolecular water of crystallization. Verification of the fact that thechange in the sample's weight was due to the water of crystallizationwas made in "(2) Structural changes".

Another sample of type I crystal of Q-35 was stored in an anhydrousatmosphere at room temperature and about 8.0% weight loss occurred inabout 700 min. When the sample was subsequently stored under atmosphericconditions, its weight increased rapidly and reverted to the same levelas the initial in about 150 min (FIG. 2). This indicates that the waterof crystallization in type I crystal of Q-35 is eliminated not only byheating but that there is also a good chance for the water ofcrystallization to be eliminated under dried conditions at roomtemperature.

Thus it was verified that dehydration of two molecules also occurredduring storage under dried conditions at room temperature and thatcomplete reabsorption of water was achieved by the dehydrate when it wasstored under atmospheric conditions at 40-50% R.H. One may then ask whatwill be the state in which type I crystal of Q-35 exists at low humidityin the presence of a very small amount of water. Two of thepossibilities that can be assumed are as follows: 1) the bimolecularwater of crystallization is incorporated into the crystal even at lowhumidity and the crystal exists as a dihydrate; or 2) at humiditieslower than a certain point, the crystal exists in an intermediate statesuch as anhydride or monohydrate. To check which of the possibilitieswas real, the inventors first dehydrated the crystal, then allowedhumidified air (6% R.H.) to flow at room temperature and measured theresulting change in weight. It was verified that the crystal absorbedwater rapidly in spite of the low humidity of the atmosphere andreverted to the weight of the dihydrate in about 60 min with nointermediate state such as monohydrate being observed in the process ofwater absorption (FIG. 3). The rate of water absorption was faster at 6%R.H. than under atmospheric conditions probably due to the difference inair flow during measurements.

In the case of dehydration by heating, the TG curve (FIG. 4) was suchthat as soon as temperature rose, a gradual weight loss occurred,followed by a noticeable abrupt weight loss until a plateau was reached.In the meantime, the DTA curve had two noticeable peaks in the processof dehydration, one being a mild peak of DTA during the gradual weightloss in TG and the other being a large peak of DTA during the abruptweight loss in TG. This may be explained as follows: of the two kinds ofwater that are present, the easy to eliminate water evaporates first andthe difficult to eliminate water evaporates thereafter and these tworeaction stages combine together. As on the DTA curve, two peaks wereobserved on the DSC curve in the process of dehydration (FIG. 5). On theother hand, the TG curve (FIG. 2) as obtained with N₂ gas being allowedto flow at room temperature (under dried conditions at room temperature)was such that a moderate weight loss occurred immediately after N₂ gaswas allowed to flow, followed by a gradual weight loss which, in turn,was followed by an abrupt weight loss until a plateau was reached. Inthis case, the water in the surface of the sample evaporated first andsubsequently, as in the case of heating, the easy to eliminate waterevaporated first and the difficult to eliminate water evaporatedthereafter and these two reaction stages would have combined together.

(2) Structural Changes

1 Infrared Absorption Spectroscopy

i) Heating (80° C.) Followed by Standing in an Indoor Atmosphere

It was verified by the TG method that type I crystal of Q-35, whenheated (80° C.), experienced a weight loss corresponding to thetheoretical value for the water of crystallization and that subsequentcooling to room temperature caused reversion to the initial weight.Since the amount of the change in weight agreed with the theoreticalvalue for the water of crystallization, the inventors estimated that theweight change of interest was due to the desorption of the two moleculesof the water of crystallization and verified this assumption by infraredabsorption spectroscopy.

The spectrum for the initial state showed a strong ν_(O--H) (H₂ O) peakdue to the water of crystallization (FIG. 6). Upon heating (80° C.), theabsorption of ν_(O--H) (H₂ O) disappeared completely, verifying that thecrystal dehydrated at 80° C. to become an anhydride (FIG. 6). Thespectrum also changed at smaller wave numbers than ν_(C)═O (carboxylateand ketone: 1622 cm⁻¹), suggesting that a certain change occurred as aresult of dehydration. The water of crystallization was bound to theoxygen in carboxylate (Q-35 assuming the betaine structure) and theν_(C)═O absorption by carboxylate (at 1622 and 1459 cm⁻¹) showed slightchanges in the shape of peaks. Subsequently, the crystal was cooledunder dried conditions and stored at room temperature but there was nonoticeable absorption of ν_(O--H) (H₂ O) and the spectrum agreed withthat obtained after heating, showing that the crystal remained in adehydrated state (FIG. 7). However, upon storage under atmosphericconditions, absorption of ν_(O--H) (H₂ O) comparable to the one observedin the initial state occurred in about 24 h and the other peaks were incomplete agreement with those in the initial spectrum, showing that thecrystal assumed the same molecular structure of dihydrate as in theinitial state (FIG. 8), whereby it was verified that the dehydrateincorporated water in the presence of water at room temperature. Theseresults show the following: upon heating, type I crystal of Q-35 had thewater of crystallization eliminated to become an anhydride but whenstored under atmospheric conditions, the crystal absorbed water torevert to the same molecular structure of dihydrate as in the initialstate.

ii) Exposure to an Anhydrous Atmosphere at Room Temperature, Followed byStanding Under Atmospheric Conditions

It was verified by the TG method that as in heating, exposure to ananhydrous atmosphere at room temperature caused a weight losscorresponding to the theoretical value for the water of crystallizationand that subsequent standing under atmospheric conditions causedreversion to the initial weight. An infrared absorption spectrumverified that the weight loss due to heating was caused by dehydration.Other infrared absorption spectra were examined to confirm that theweight changes during exposure to an anhydrous atmosphere at roomtemperature were due to water and to check whether the dehydrateobtained by heating had a different molecular structure from thedehydrate obtained by exposure to an anhydrous atmosphere at roomtemperature.

Upon storage under atmospheric conditions, the absorption of ν_(O--H)(H₂ O) disappeared as in heating and the resulting spectrum agreedcompletely with that obtained upon heating (FIG. 9); this indicated thatmerely by storing it under dried conditions at room temperature, thecrystal was dehydrated to become an anhydride, which took on the samemolecular structure as that obtained by heating. Upon subsequent storageunder atmospheric conditions, the absorption of ν_(O--H) (H₂ O)comparable to that obtained in the initial state was observed as inheating and the resulting spectrum agreed with the initial one,verifying that the crystal assumed the same molecular structure ofdihydride as in the initial state (FIG. 10). It was therefore verifiedthat upon storage under dried conditions at room temperature, thecrystal was dehydrated to become an anhydride, that the dehydratedanhydride had the same molecular structure as the dehydrated anhydridethat was formed by heating, and that upon subsequent storage underatmospheric conditions, the crystal reverted to the same molecularstructure of dihydride as in the initial state.

Measurements of Infrared absorption spectra thus verified the following:type I crystal of Q-35 was dehydrated to yield an anhydride both underheating (80° C.) and upon exposure to an anhydrous atmosphere at roomtemperature; the dehydrated product had the same molecular structureirrespective of the drying conditions; and upon storage in a roomtemperature atmosphere, the crystal absorbed moisture again to revertthe same molecular structure of dihydrate as in the initial state,indicating the reversible nature of water desorption.

(2) Powder X-ray Diffraction

i) Heating (80° C.) Followed by Standing Under Atmospheric Conditions

It was verified by infrared absorption spectra that dehydration occurredboth under heating and upon exposure to an anhydrous atmosphere at roomtemperature but that water returned upon storage under atmosphericconditions. Under the circumstances, the changes that occurred in thecrystal structure as a result of dehydration were examined by powderX-ray diffraction.

The spectrum for the initial state is shown in FIG. 11. Upon heating(80° C.), the large peak that occurred at 24.2° in the initial statedisappeared and other aspects of the spectrum varied to produce anentirely different spectrum (FIG. 12). This result, taken in combinationwith the verification by an infrared absorption spectrum thatdehydration was caused by heating, means that when dehydration occurredupon heating, not only was the molecule of water eliminated but also thecrystal itself took on a different structure. When the crystal wassubsequently cooled to room temperature under dried conditions,examination by an infrared absorption spectrum showed that the crystalretained the dehydrated state even when it was first cooled under driedconditions, then stored at room temperature. Powder X-ray diffractionalso yielded a spectrum in agreement with the one obtained by heating(dehydration) showing the retention of the crystal structure of thedehydrated product (FIG. 13), with the crystal structure being the sameas that of the dehydrated product obtained by heating. However, uponstorage under atmospheric conditions, the large peak that occurred at24.2° in the initial state appeared again in 14 h, yielding a spectrumin complete agreement with the initial spectrum (FIG. 14). It wasalready verified by an infrared absorption spectrum that upon storageunder atmospheric conditions, water returned to have the crystal revertto the initial molecular structure. Now it was verified by powder X-raydiffraction, too, that upon storage under atmospheric conditions, thecrystal structure of the dehydrated product reverted to the initialstate, namely, the structure having two molecules of the water ofcrystallization. Combining the results of infrared absorptionspectroscopy with those of powder X-ray diffraction, one can see thatheating caused dehydration which, in turn, caused changes in the crystalstructure but that upon storage under atmospheric conditions, waterreturned while, at the same time, the crystal structure also reverted tothe initial state. Desorption of water was reversible and the hydrateand the dehydrate had different crystal structures and desorption ofwater was accompanied by a simultaneous change in the crystal structure,which change in the crystal structure was also reversible.

ii) Exposure to an Anhydrous Atmosphere at Room Temperature, Followed byStanding Under Atmospheric Conditions

Infrared absorption spectroscopy revealed dehydration even upon exposureto an anhydrous atmosphere at room temperature and the change exhibiteda similar behavior to the one that accompanied heating. Hence,examination was made in order to check whether a similar behavior wouldbe exhibited in powder X-ray diffraction.

Upon storage under dried conditions, the spectrum changed over timeuntil it agreed with the FIG. 15 spectrum after heating (dehydration) asshown in FIG. 16. Since it was already verified by an infraredabsorption spectrum that dehydration occurred upon exposure to ananhydrous atmosphere at room temperature, the sample that had beenstored under dried conditions for powder X-ray diffraction was adehydrated product. The dehydrate after storage under dried conditionsshowed the same molecular structure in infrared absorption spectrumwhether it had been heated or stored under dried conditions at roomtemperature. Similarly, said dehydrate was verified to have the samecrystal structure as the dehydrate formed by heating. Upon storage underatmospheric conditions, the crystal produced a spectrum in 2 h thatagreed completely with the FIG. 18 powder X-ray diffraction spectrum (asshown in FIG. 17). Infrared absorption spectroscopy showed that waterreturned to the crystal when it was stored under atmospheric conditionsand powder X-ray diffraction also verified that as in the case of thechanges due to heating, the sample reverted to the initial crystalstructure of dihydrate.

These results showed the following: type I crystal of Q-35 dehydrated tobecome an anhydride either by heating or upon storage under driedconditions at room temperature and since the two dehydrated productsassumed the same molecular and crystal structures, they were identicalsubstances; when the dehydrates were stored under atmosphericconditions, they reverted to identical substances that took on the samemolecular and crystal structures of dihydrate as in the initial state.

(3) Single-crystal X-ray Analysis

Infrared absorption spectroscopy and powder X-ray diffraction revealedthat dehydration occurred under both heating and exposure to ananhydrous atmosphere at room temperature, with the dehydrated productsassuming identical molecular and crystal structures; it was also foundthat upon storage under atmospheric conditions, the dehydrates revertedto the same molecular and crystal structures of dihydrate as in theinitial state. To further support these facts, the inventors conductedsingle-crystal X-ray analyses.

Samples of type I crystal of Q-35 were prepared for use insingle-crystal X-ray analysis. On the basis of measurements conducted onthese samples, a composite spectrum for powder X-ray diffraction wasconstructed and this was found to agree with the powder X-raydiffraction spectrum (FIG. 19) obtained upon standing under atmosphericconditions (as shown in FIG. 20). Subsequently, samples of type Icrystal of Q-35 were also prepared for use in single-crystal X-rayanalysis by drying an anhydrous atmosphere at room temperature. On thebasis of measurements conducted on those samples, a composite spectrumfor powder X-ray diffraction was constructed and this was found to agreewith the FIG. 21 X-ray diffraction spectrum obtained by heating andsubsequent exposure to an anhydrous atmosphere at room temperature (asshown in FIG. 22). Hence, it was verified that the dried single crystalhad been dehydrated. The dehydrated single crystal had experienced achange in lattice constant (initial: b=12.966 (2) Å; dehydrated crystal:b=38.34 (2) Å; no change in a, c and β), with the resulting change inthe structure of the trimer.

Crystal structural diagrams for the initial state are shown in FIGS. 23and 24; crystal structural diagrams for the dehydrated product are shownin FIGS. 25 and 26. The crystal that had been dried under driedconditions at room temperature was stored under atmospheric conditionsand subjected to measurements again; the sample was verified to have thesame crystal structure as in the initial state (FIGS. 27 and 28).

Powder X-ray diffraction showed that the hydrate and the dehydrate haddifferent crystal structures and the structural change involved wasreversible; these results were also supported by single-crystal X-rayanalysis.

5) Conclusion

The foregoing experimental results made the following points clear aboutthe behavior of the water of crystallization in type I crystal of Q-35.

By heating or storage under dried conditions at room temperature,dehydration as accompanied by changes in the crystal structure occurredto produce anhydrides.

Irrespective of the drying conditions employed as to whether it was byheating or by storage under dried conditions at room temperature, thedehydrated products had the same molecular and crystal structures.

The amount of dehydration agreed quantitatively with the theoreticalvalue of water content in the dehydrate.

Upon storage under atmospheric conditions, the dehydrate absorbedmoisture in air to revert to type I crystal of Q-35.

Desorption of water was reversible.

The amount of water absorption by the dehydrates agreed quantitativelywith the theoretical value for two molecules of water.

The dehydrates changed to type I crystal of Q-35 in the presence of theslightest amount of water in the atmosphere it is placed; hence, thewater of crystallization in type I crystal of Q-35 is stable as long asit is handled in the usual manner.

As already mentioned herein, type III crystal of Q-35 has very poorstability. In contrast, type I crystal (dihydrate) and type II(monohydrate) of Q-35, both of which dehydrate under drying conditionsto become anhydrides, have been verified to absorb moisture in air againupon storage under atmospheric conditions to revert to the initial typeI and type II crystals, respectively, of Q-35. Under the circumstances,tests were conducted to compare the stability of the two types ofcrystal by the methods described below, giving the results alsodescribed below.

Test 1--Moisture Absorption Test

Type I crystal and type II crystal of Q-35 were each placed at 40° C.and left to stand under varying humidity conditions of 0% R.H., 52.4%R.H., 75% R.H. and 100% R.H. to investigate the weight changes thatoccurred 4-7 days later. The results are shown in Table 1.

                  TABLE 1    ______________________________________    Weight changes at 40° C. and at varying    humidified conditions             Sample                   Days (%)             (mg)  4       5        6     7    ______________________________________    (Type II crystal)    0% R.H.    113.0   -2.57   -2.48  -2.12 -2.48    52.4% R.H. 129.1   0.31    -0.15  -0.08 -0.15    75% R.H.   113.0   0.53    0.53   0.62  0.62    100% R.H.  118.9   3.78    4.46   4.46  4.71    (Type I crystal)    0% R.H.    127.9   -4.53   -8.29  -8.21 -8.05    52.4% R.H. 132.5   0.38    0.23   0.30  0.53    75% R.H.   192.5   0.42    0.47   0.26  0.42    100% R.H.  129.1   0.70    0.39   0.39  0.34    ______________________________________

Type II crystal experienced a little more than 2% weight loss at 0% R.H.but its weight change was no more than 1% at 52.4% R.H. and 75% R.H.However, its weight increased by about 5% at 100% R.H. On the otherhand, type I crystal experienced about 8% weight loss at 0% R.H. but thechange was within 1% at all other relative humidities. At lowerhumidities, type I crystal would lose the water of crystallization.

When type II crystal was stored at 40° C. for 1 week at 0% R.H. and 75%R.H., the resulting powder X-ray diffraction spectra (FIGS. 29a and 29b)both agreed with the initial spectrum for type II crystal; however, thespectrum (FIG. 29c) obtained after storage at 40° C. for 1 week at 100%R.H. did not agree with the initial spectrum for type II crystal and wasestimated to be a mixture of spectra for diffraction peaks of type I andtype III crystals.

On the other hand, the powder X-ray diffraction spectrum (FIG. 30) fortype I crystal that was stored at 40° C. for 1 week at 100% R.H. agreedwith the initial spectrum for type I crystal.

From these results, one may well conclude that type I crystal is moreadvantageous than type II crystal in terms of pharmaceuticalsmanufacture for the reason that although it experiences a weight changeat 0% R.H. (under drying conditions) on account of the loss of the waterof crystallization, type I crystal, when placed at high humidities, doesnot exhibit any marked moisture absorption, nor does it involve anycrystal dislocation.

Test 2--Effects of Blending

Q-35, when it is to be used as a medicine, is held to be suitablyformulated as an oral preparation of 100-200 mg. Therefore, theformulation would have a high content of active ingredient, presenting astrong need for performing wet granulation. Hence, in simulated wetgranulation, blending was done in water and/or ethanol to check whetherthe crystal form would change or not; to this end, each of type II and Icrystals was blended in ethanol, 50% aqueous ethanol solution or waterand, thereafter, powder X-ray diffraction spectra were measured.

The powder of type II crystal, when blended in ethanol, provided apowder X-ray diffraction spectrum (FIG. 31a) that agreed with the onefor the initial type II crystal, thus showing that there was no changein the crystal form. However, when blended in 50% aqueous ethanolsolution or water, the powder gave a mixture of diffraction peaks fortype II crystal and type I crystal (FIGS. 31b and 31c). It was thusverified that type II crystal, when blended using a solvent with no morethan 50% ethanol content, shifted partially to type I crystal.

On the other hand, the powder of type I crystal produced powder X-raydiffraction spectra that agreed with the one for the initial stage oftype I crystal irrespective of the solvent in which it was blended (FIG.32). It was thus verified that the blending of type I crystal did notcause any shifting therefrom.

Therefore, type I crystal was found to be more desirable than type IIcrystal in pharmaceutical formulation procedures by wet granulation.

Industrial Applicability

As described on the foregoing pages, type I crystal of Q-35 according tothe invention exhibits excellent stability under various conditions suchas moisture absorption and blending in solvents and, hence, it is a mostadvantageous crystal form in pharmaceutical formulation procedures.

We claim: 1.1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid dihydrate having the following formula: ##STR4##
 2. Apharmaceutical composition comprising an effective amount of thecompound according to claim 1 and a pharmaceutically acceptable carrier.3.1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid dihydrate which has been obtained by recrystallizing1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3-carboxylicacid from a 50:50 mixture of water and ethanol.